Gene regulatory region that promotes early seed-specific transcription

ABSTRACT

Nucleic acid sequence capable of regulating transcription during embryogenesis in plants is provided. This sequence may be used in transgenic plants to promote high levels of expression of foreign and endogenous genes in developing seeds to affect seed lipid metabolism, protein or carbohydrate composition and accumulation, or seed development. In addition, these sequences may be useful for the production of modified seed containing novel recombinant proteins which have pharmaceutical, industrial or nutritional value, or novel products like plastics.

[0001] This application derives priority from U.S. Provisional PatentApplication No. 60/206,787, which was filed May 24, 2000.

FIELD OF THE INVENTION

[0002] This invention relates to a nucleic acid sequence, whichregulates transcription during embryogenesis in plants. Morespecifically, the nucleic acid sequence of the present invention can beused in transgenic plants to promote high levels of expression offoreign and endogenous genes in developing seeds to affect seed lipidmetabolism, protein or carbohydrate composition and accumulation, orseed development. In addition, the nucleic acid sequence of the presentinvention can be useful for the production of modified seed containingnovel recombinant proteins which have pharmaceutical, industrial ornutritional value, or novel products like plastics.

BACKGROUND

[0003] Most of the information about seed-specific gene expression comesfrom studies of genes encoding seed storage proteins like napin, a majorprotein in the seeds of Brassica napus, or conglycinin of soybean.Furthermore, upstream DNA sequences directing strong embryo-specificexpression of these storage proteins have been used successfully intransgenic plants to manipulate seed lipid composition and accumulation(Voelker et al., 1996). However, expression of storage protein genesbegins fairly late in embryogenesis. Thus, promoters of seed storageprotein genes may not be ideal for all seed-specific applications. Forexample, storage oil accumulation commences significantly before thehighest level of expression of either napin (Stalberg et al., 1996) orconglycinin (Chen et al., 1988) is achieved. It is, therefore ofinterest to identify other promoters which control expression of genesin developing embryos with temporal specificity different from that ofseed storage proteins.

SUMMARY OF TE INVENTION

[0004] The nucleic acid sequence of the present invention can be used toregulate transcription during embryogenesis in plants. By the presentinvention it is possible to promote high levels of expression of foreignand endogenous genes in developing seeds to affect seed lipidmetabolism, protein or carbohydrate composition and accumulation, orseed development. The present invention can also be useful for theproduction of modified seed, which contains novel recombinant proteins.

BRIEF DESCRIPTION OF THE DRAWING

[0005] The FIGURE shows nucleic acid sequence of the insert in theplasmid pLfKCS3-GUS.

DETAILED DESCRIPTION

[0006] The inventors have determined that a more suitable generegulatory region for directing gene expression aimed at seed oilmodification would originate from a seed lipid metabolic gene expressedin a seed-specific manner. One such gene is LfKCS3, which encodes acondensing enzyme of very long chain fatty acid biosynthesis inLesquerella fendleri. LfKCS3 condensing enzyme is thought to belocalized in the endoplasmic reticulum where it catalyzes the sequentialelongation of C18 fatty acyl chains to C20 in length. RNA blot analysesshowed that the LfKCS3 gene transcript was present only in developingembryos. The inventors isolated the 5′ regulatory region of the LfKCS3gene and in the present application demonstrate that it is useful inpromoting early seed-specific transcription of heterologous genes inArabidopsis. Regulatory 5′ DNA sequences promoting early seed-specifictranscription found upstream of other plant KCS genes have also beenisolated and disclosed previously (U.S. Provisional Patent Applicationfiled Aug. 4, 1999, Inventors Kunst and Clemens).

[0007] Isolated transcription regulatory region from the LpfKCS3 gene iscapable of directing expression of desired genes at an early stage ofdevelopment in a seed-specific manner. Because this regulatory sequencecan also promote transcription in developing seeds of a different plantspecies, it can be used in a variety of dicotyledonous plants formodification of the seed phenotype.

[0008] Examples of applications wherein the nucleic acid sequence of thepresent invention can be useful include, for example:

[0009] (1) altered seed fatty acid compositionor seed oil compositionand accumulation,

[0010] (2) altered seed protein or carbohydrate composition oraccumulation,

[0011] (3) enhanced production of desirable seed products,

[0012] (4) suppression of production of undesirable seed products usingantisense, co suppression or ribozyme technologies,

[0013] (5) production of novel recombinant proteins for pharmaceutical,industrial or nutritional purposes,

[0014] (6) production of novel compounds/products in the seed, ie.secondary metabolites, plastics, etc.

[0015] The methods employed in the isolation of the nucleic acid of thepresent invention and the uses thereof are discussed in the followingnon-limiting examples:

EXAMPLES

[0016] Isolation of a Seed-Specific Promoter Region form Lesquerellafendleri

[0017] A Lesquerella fendleri genomic DNA library was obtained from Dr.Chris Somerville, Carnegie Institution of Washington, Stanford, Calif.The genomic library was plated on E. coli LE392 (Promega) and about150,000 clones were screened using Arabidopsis FAE1 gene (James et al.,1995) as a probe. The probe was prepared by PCR using pGEM-7Zf(+)-FAE1(Millar and Kunst, 1997) as a template with FAEE upstream primer,5′-CCGAGCTCAAAGAGGATACATAC-3′ and FAE1 downstream primer,5′-GATACTCGAGAACGTTGGCACTCAGATAC-3′. PCR was performed in a 100 reactioncontaining 10 ng of the template, 2 mM MgCl₂, 1.1 μM of each primer, 100μM of (dCTP+dGTP+dTTP) mix, 50 μCi of [α-32P]dATP, 1× PCR buffer and 2.5units of Taq DNA polymerase (Life Technologies). Amplificationconditions were: 2 min of initial denaturation at 94° C., 30 cycles of94° C. for 15 sec, 55° C. for 30 sec, 72° C. for 1 min and 40 sec,followed by a final extension at 72° C. for 7 min. The amplifiedradiolabeled probe was purified by QIAquick PCR Purification Kit(Qiagen) and denatured by boiling before adding to the hybridizationsolution Hybridization took place overnight at 65° C. in a solutioncontaining 6× SSC, 20 mM NaH₂PO₄. 0.4% SDS, 5× Denhardt's solution, and50 μg/ml sonicated, denatured salmon sperm DNA (Sigma) and washing wasperformed three times for 20 min each in 2× SSC, 0.5% (w/v) SDS at 65°C.

[0018] Nine clones with sequences corresponding to the Arabidopsis FAE1gene were isolated from the Lesquerella fendleri genomic library. Thephage DNA from those nine clones was extracted and purified using QIAGENLambda Mini Kit (Qiagen) according to the manufacturer's protocol. Oneof them was digested with EcoRI and a 4.3 kb fragment was subcloned intothe pGEM-7Zf(+) vector (Promega) cut with EcoRI, resulting in the vectorpMHS15. The whole insert was sequenced with ABI automatic 373 DNAsequencer using fluorescent dye terminators. Approximately 573 bp of the5′ upstream region of the 4.3 kb genomic DNA was amplified using thehigh fidelity Pfu polymerase (Stratagene) with a forward primer5′-CGCAAGCTTGAATTCGGAAATGGGCCAAG-3′ and a reverse primer5′-CGCGTCGACTG=TGAGTTTGTGTCGGG-3′. The amplified fragment was insertedupstream of the GUS gene in pBI101 (Clontech) cut with HindIII and SalI,resulting in the vector pLfKCS3-GUS. The sequence of the insert in theplasmid pLfKCS3-GUS is shown in FIG. 1.

[0019] Functional Analysis of the LfKCS3 5′ Upstream Region

[0020] To evaluate the ability of the 5′ upstream fragment of the LfKCS3gene to confer seed-specific and temporal regulation of gene expressionin plants, the pLfKCS3-GUS construct was introduced into Agrobacteriumtumefaciens strain GV3101 (MP90) (Koncz and Schell, 1986) by heat-shockand selected for resistance to kanamycin (50 μg/mL). A. thaliana ecotypeColumbia was transformed with A. tumefaciens harbouring the pLfKCS3-GUSconstruct using floral dip method (Clough and Bent, 1998). Screening fortransformed seed was done on 501 g/mL kanamycin as described previously(Katavic et al., 1994). Approximately 100 transgenic lines weregenerated for each construct.

[0021] Histochemical localization of GUS activity in transgenic plantswas done on tissue sections as follows. Sections were incubated in 50 mMsodium phosphate, pH 7.0, 0.5 mM potassium ferricyanide, 0.5 mMpotassium ferrocyanide, 10 mM EDTA, 0.05%(w/v) triton X-100, and 0.35mg/ml 5-bromo4-chloro-3-indolyl-p-D-glucuronide (X-Gluc) for 4 to 7hours at 37° C. (Jefferson, 1987). Following staining the blue-stainedsamples were fixed in 70°/ethanol.

[0022] Using this assay, over 30 independent transgenic Arabidopsislines were examined for the embryo-specific expression of the GUS gene.In addition, leaves, stems, inflorescences, roots, and siliques atdifferent stages of development were histochemically stained forp-glucuronidase activity. The GUS reporter gene fused to the LfKCS3promoter was not expressed in any of the vegetative tissues, whereas itwas highly expressed in developing embryos. We also compared the LfKCS3promoter with the LFAH12 promoter that was reported to be an early andseed-specific promoter active already at the torpedo stage ofArabidopsis (Broun et al., 1998). Our results suggest that the LfKCS3promoter is active even earlier. Thus, the onset of the LfKCS3 promoteractivity coincides with or precedes that of storage oil accumulation.GUS activity in all the examined transgenic lines persisted throughoutsubsequent embryo development. Thus the LfKCS3 promoter is useful forseed-specific expression of foreign genes in transgenic plants.

[0023] In conclusion, we have demonstrated that the elements whichconfer both tissue specific and developmental regulation of a reportergene linked to the LfKCS3 promoter reside within the 573 bp upstream ofthe AUG translation initiation codon. Our experiments also show that theLesquerell afenaleri LfKCS3 promoter directs seed-specific expression atleast as early as the torpedo stage embryo and that the it is capable ofpromoting transcription in plants other than Lesquerell afendleri.

[0024] It should also be mentioned that the seed-specific expressionconferred by the LfKCS3 promoter is independent of the native terminatorat the LfKCS3 gene 3′ end. In all our constructs, a terminator derivedfrom the Agrobacterium nopaline synthase gene was used. Thus, thesequence in the 573 bp promoter construct is sufficient for the desiredexpression profile independent of ancillary sequences.

REFERENCES

[0025] Broun, P., Boddupalli, S., and Somerville, C. (1998) Abifunctional oleate 12-hydroxylase: desaturase from Lesquerellafendleri. Plant J. 13, 201-210

[0026] Chen, Z. L., Pan, N. S., and Beachy, R. N. (1988) A DNA sequenceelement that confers seed-specific enhancement to a constitutivepromoter. EMBO J. 6: 3559-3564.

[0027] Clough, S. J. and Bent, A. F. (1998) Floral dip: a simplifiedmethod for Agrobacterium-mediated transformation of Arabdiopsisthaliana. Plant J. 16: 735-743.

[0028] James, D. W., Jr., Lim, E., Keller, J., Plooy, I., Ralston, E.,and Dooner, H. K. (1995) Directed tagging of the Arabidopsis FATTY ACIDELONGATION (FAE1) gene with the maize transposon Activator. Plant Cell7: 309-319.

[0029] Jefferson, R. A., Kavanaugh, T. and Bevan, M. W. (1987) GUSfusions: β-glucuronidase as a sensitive and versatile gene fusion markersystem in higher plants. EMBO J. 6: 3901-3907.

[0030] Katavic, V., Haughn, G. W., Reed, D., Martin, M., and Kunst, L.(1994) In planta transformation of Arabidopsis thaliana Mol. Gen. Genet.245: 363-370.

[0031] Koncz, C. and Schell, J. (1986) The promoter of T_(L)-DNA gene Scontrols the tissue-specific expression of chimaeric genes carried by anovel type of Agrobacterium binary vector. Mol. Gen. Genet. 204:383-396.

[0032] Stalberg, K., Ellerstoem, M., Ezcurra, I., Ablov, S., and Rask,L. (1996) Disruption of an overlapping e-box-ABRB motif abolished hightranscription of the napA storage-protein promoter in transgenicBrassica napus seeds. Planta 199: 515-519.

[0033] Voelker, T. A., Hayes, T. R., Cranmer, A. M., Turner, J. C., andDavies H. M. (1996) Genetic engineering of a quantitative trait:Metabolic and genetic parameters influencing the accumulation of lauratein rapeseed. Plant J. 9: 229-241.

1 5 1 588 DNA Lesquerella fendleri 1 gaattcggaa atgggccaag tgaaatggaaatagagcttc aatccattta gtcccactca 60 aaatggtgct cgaattatat ttagttacgttcgaatcaga caaccaagta tttggttaat 120 aaaaaccact cgcaacaaag gaaaaacaccaagcgcgtgc gtccaacatc cgacggaagg 180 ggggtaatgt ggtccgaaaa ccttacaaaaatctgacgtc atctaccccc gaaaacgttg 240 aatcgtcaac gggggtagtt ttcgaattatctttttttta ggggcagttt tattaatttg 300 ctctagaaat tttatgattt taattaaaaaaagaaaaaga atatttgtat atttattttt 360 tatactcttt ttttgtccaa ctatttctcttattttggca actttaacta gactagtaac 420 ttatgtcaat gtgtatggat gcatgagagtgagtatacac atgtctaaat gcatgcctta 480 tgaaagcaac gcaccacaaa acgaagacccctttacaaat acatctcatc ccttagtacc 540 ctcttactac tgtcccgaca caaactcaaaacaatgacat ctctaaac 588 2 23 DNA Artificial Sequence FAE1 PCR Primer 2ccgagctcaa agaggataca tac 23 3 29 DNA Artificial Sequence FAE1 PCRPrimer 3 gatactcgag aacgttggca ctcagatac 29 4 29 DNA Artificial SequencePCR Primer 4 cgcaagcttg aattcggaaa tgggccaag 29 5 29 DNA ArtificialSequence PCR Primer 5 cgcgtcgact gttttgagtt tgtgtcggg 29

What we claim is:
 1. An isolated nucleic acid fragment comprising anucleic acid sequence encoding a promoter for directing seed-specifictranscription of contiguous genes in plants.
 2. An isolated nucleic acidfragment according to claim 1, wherein said sequence is defined by thenucleic acid sequence of SEQ. ID. NO.
 1. 3. An isolated nucleic acidfragment according to claim 1, wherein said sequence has a sequenceidentity of 95% or greater to the nucleic acid sequence of SEQ. ID.NO.
 1. 4. An isolated nucleic acid fragment according to claim 1,wherein said sequence has a sequence identity of 85% or greater to thenucleic acid sequence of SEQ. ID. NO.
 1. 5. An isolated nucleic acidfragment according to claim 1, wherein said sequence has a sequenceidentity of 65% or greater to the nucleic acid sequence of SEQ. ID.NO.
 1. 6. An isolated nucleic acid fragment comprising a nucleic acidsequence encoding a promoter for enhancing expression of endogenous andforeign genes in seeds of plants.
 7. An isolated nucleic acid fragmentaccording to claim 3, wherein said wherein said sequence is defined bythe nucleic acid sequence of SEQ. ID. NO.
 1. 8. An isolated nucleic acidfragment according to claim 1, wherein said sequence promotes expressionof genes, which affect seed lipid metabolism, protein or carbohydratecomposition and accumulation, or seed development.
 9. An isolatednucleic acid fragment according to claim 1, wherein said genes enhancethe pharmaceutical, industrial, nutritional value or usefulness forplastic product fabrication of seed products of plants.
 10. An isolatednucleic acid fragment comprising a nucleic acid sequence encoding apromoter for enhancing production of recombinant proteins in seeds ofplants.